7-Acyl-3-(substituted tetrazolyl thiomethyl)cephalosporins

ABSTRACT

The compounds of this invention are cephalosporins having various acyl substituents at the 7-position and a substituted tetrazolyl thiomethyl group at the 3-position of the cephem nucleus and intermediates for the preparation thereof. The 7-acylated compounds have antibacterial activity.

This invention comprises a new series of cephalosporin compounds whichhave antibacterial activity when administered either orally orparenterally and to intermediates for the preparation thereof. Inparticular, the structures of the biologically active cephalosporincompounds of this invention are characterized by having a novellysubstituted tetrazolyl thiomethyl group at the 3-position of the cephemnucleus. Also, this invention extends to methods and compositions fortreating certain bacterial infections using these new compounds as wellas to certain chemical intermediates and methods for preparing thecompounds described hereafter.

The compounds of this invention are represented by the followingstructural formula: ##STR1## in which: each individual R¹ is hydrogen orlower alkyl;

n is one to ten;

R² is hydroxy, amino, lower alkylamino or di(lower)alkylamino; and

R³ is an acyl group selected from the group consisting of: ##STR2##where: X is thienyl; dihydrophenyl; phenyl; phenyl mono-substituted withhydroxy, hydroxymethyl, formamido, ureido or carboxymethylamino; or3-fluoro-4-hydroxyphenyl;

A is NH₂, OH, COOH or SO₃ H;

Y is cyano, sydnone or aminomethylphenyl;

Z is methyl, trifluoromethyl, trifluoroethyl or pyridyl; and

m is zero to two,

or a non-toxic pharmaceutically acceptable salt thereof.

As used herein, the term "lower alkyl" refers to groups having from oneto four carbon atoms.

It will be recognized that the 4-carboxylic acid group of the compoundsof Formula I may be readily esterified by methods well known to the art.These esters include, for example, simple alkyl and aryl esters as wellas esters which are easily cleaved, within the body, to the parent acidsuch as indanyl, pivaloyloxymethyl, acetoxymethyl, propionyloxymethyl,glycyloxymethyl, phenylglycyloxymethyl and thienylglycyloxymethyl estersand others. Of course, when A is COOH, this group may be similarlyesterified. All such esters are included within the scope of thisinvention.

Preferred compounds of this invention are represented by Formula I whereR¹ is hydrogen; n is one to five; R² is hydroxy, amino, lower alkylaminoor di(lower)alkylamino; R³ ##STR3## X is thienyl, dihydrophenyl, phenyl,phenyl mono-substituted with hydroxy, hydroxymethyl, formamido, ureido,carboxymethylamino, or 3-fluoro-4-hydroxyphenyl; A is NH₂, OH, COOH orSO₃ H; Y is cyano, sydnone or aminomethylphenyl; Z is methyl,trifluoromethyl, trifluoroethyl or pyridyl and m is zero to two.

Advantageous compounds of this invention are represented by Formula Iwhere R¹ is hydrogen; n is one to five; R² is hydroxy, amino, loweralkylamino or di(lower)alkylamino; R³ is ##STR4## X is phenyl orhydroxyphenyl; A is NH₂ or OH; Z is methyl, trifluoromethyl ortrifluoroethyl and m is zero to two.

Most advantageous are the compounds represented by Formula I where R¹ ishydrogen; n is one to five; R² is hydroxy or amino; R³ is ##STR5## X isphenyl or 4-hydroxyphenyl; A is NH₂ or OH; Z is trifluoromethyl and m iszero.

Examples of the most preferred 7-acyl substituents (R³ NH--) of thecompounds of Formula I are listed below:

α-hydroxyphenylacetamido

α-aminophenylacetamido

α-amino-4-hydroxyphenylacetamido

trifluoromethylmercaptoacetamido

methylmercaptoacetamido

2,2,2-trifluoroethylsulfinylacetamido

cyanoacetamido

α-carboxythienylacetamido

α-carboxyphenylacetamido

α-sulphophenylacetamido

methylsulfonylacetamido

α-amino-4-carboxymethylaminophenylacetamido

α-amino-3-fluoro-4-hydroxyphenylacetamido

3-sydnoneacetamido

4-pyridylthioacetamido

2-aminomethylphenylacetamido.

Most preferred substituted tetrazolyl groups are the following:

1-carboxymethyltetrazolyl

1-carbamoylmethyltetrazolyl

1-(2-carboxyethyl)tetrazolyl

1-(2-carbamoylethyl)tetrazolyl

1-(3-carboxypropyl)tetrazolyl

1-(3-carbamoylpropyl)tetrazolyl

1-(4-carboxybutyl)tetrazolyl

1-(4-carbamoylbutyl)tetrazolyl

1-(5-carboxypentyl)tetrazolyl

1-(5-carbamoylpentyl)tetrazolyl.

Particularly preferred are the compounds7-D-mandelamido-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7-D-mandelamido-3-(1-carbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7-D-mandelamido-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid,7-D-mandelamido-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid,7-D-mandelamido-3-[1-(3-carbamoylpropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid,7-D-mandelamido-3-[1-(3-carboxypropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid,7-D-mandelamido-3-[1-(5-carboxypentyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid,7-D-mandelamido-3-[1-(5-carbamoylpentyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid,7-(D-α-amino-4-hydroxyphenylacetamido)-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7-(D-α-amino-4-hydroxyphenylacetamido)-3-(1-carbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7-trifluoromethylmercaptoacetamido-3-(1-carbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7-trifluoromethylmercaptoacetamido-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylic acid,7-trifluoromethylmercaptoacetamido-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7-trifluoromethylmercaptoacetamido-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid and7-trifluoromethylmercaptoacetamido-3-[1-(3-carbamoylpropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

Cephalosporin derivatives having 7-acyl substituents as defined aboveare all documented in the prior art. Substitution by a substitutedS-heterocyclicthiomethyl group (--CH₂ SHet) at the 3-position of thecephem nucleus is also known and is disclosed in Netherlands Pat. No.6916151 where Het is, among others, tetrazolyl substituted with, interalia, carboxy, carbalkoxy, alkoxyalkylaminocarbonyl anddialkylaminoalkylaminocarbonyl and in Japanese Pat. No. 7205550 whereHet includes tetrazolyl substituted with --(CH₂)_(n) R³ where n is 0 to3 and R³ includes alkoxycarbonyl, carboxy, N-alkoxyalkylcarbamoyl anddialkylamino. The compounds disclosed in Netherlands Pat. No. 6916151,however, have a similarly substituted heterocyclic acetamido group atthe 7-position, while those of Japanese Pat. No. 7205550 contain a7-thienylacetamido or 7-tetrazolylacetamido group. Recently issued U.S.Pat. No. 3,819,623 discloses but does not claim cephalosporins bearing a7-heterocyclicacetamido or 7-heterocyclicthioalkylacetamido group andhaving in the 3-position, inter alia, thiomethyltetrazolyl substitutedwith carboxy, alkoxycarbonyl, carboxyalkyl, alkoxycarbonylalkyl anddialkylaminoalkylaminocarbonylalkyl. No compounds containing bothapplicant's 7-acyl groups and the 3-substituted tetrazolylthiomethylmoiety disclosed herein are believed to be known to the art.

The compounds of Formula I are prepared by acylation of an appropriate7-amino-3-substituted tetrazolylthiomethyl cephalosporin nucleus ofFormula II: ##STR6## in which:

each individual R¹ is hydrogen or lower alkyl;

n is one to ten;

R² is hydroxy, amino, lower alkylamino or di(lower)alkylamino; and

R⁴ is hydrogen or a protecting ester group, with an appropriateacylating agent followed by removal of the protective groups whenpresent. The carboxylic acid group of the acylating agent is activatedby any of the standard methods such as conversion to the mixedanhydride, acid chloride or activated ester. In addition, a reagent suchas dicyclohexylcarbodiimide or carbonyldiimidazole can be used providedthat the carboxyl group on the cephem nucleus is protected with aneasily removable protecting group such as a benzhydryl, t-butyl,trichloroethyl, benzyl, benzyloxymethyl, p-nitrophenyl, p-methoxyphenyl,p-methoxybenzyl or p-nitrobenzyl ester. When A is NH₂, the α-amino groupof the acylating agent is, preferably, protected prior to acylation withan easily removable protective group known in the art such ast-butoxycarbonyl, trichloroethoxycarbonyl, benzyloxycarbonyl, the methylacetoacetate adduct or similar groups commonly used in the synthesis ofpeptides. The compounds represented by Formula II above are alsoconsidered as objects of this invention.

Alternatively, the compounds of Formula I are prepared by acylating7-aminocephalosporanic acid with an appropriately protected acylatingagent, as described above, and then displacing the 3-acetoxy group withthe desired substituted tetrazole thiol with subsequent removal of theprotective group(s). The substituted tetrazole thiols of the formula:##STR7## in which:

each individual R¹ is hydrogen or lower alkyl;

n is one to ten; and

R² is hydroxy, amino, lower alkylamino or di(lower)alkylamino,

are also objects of this invention.

The protective groups can be removed according to methods well known tothe art, such as with trifluoroacetic acid when t-butyl ort-butoxycarbonyl protective groups are used. The resulting salt isconverted to the zwitterionic product or to the free acid by means of abasic ion exchange resin such as polystyrene-amine ion exchange resin(Amberlite IR-45) or else by basification of an aqueous solution of thesalt.

The acylating agents used as starting materials are either known orprepared by known methods.

The 7-amino-3-substituted tetrazolylthiomethyl cephalosporin startingmaterials of Formula II are prepared from reaction of7-aminocephalosporanic acid and a substituted tetrazole thiol of FormulaIII.

The substituted tetrazole thiols of Formula III where R² is hydroxy areprepared by reaction of an isothiocyanate, for example ethylisothiocyanoacetate, or an N-alkyl dithiocarbamate, such as methyl2-carboxyethyldithiocarbamate, with an azide such as sodium azide. WhenR² is amino, lower alkylamino or di(lower)alkylamino, the tetrazolethiols of Formula III are prepared from the corresponding tetrazolethiols where R² is hydroxy by standard methods for the preparation ofamides from acids, for example, by reaction of a tetrazole thiol whereR² is hydroxy with 1,1-carbonyldiimidazole and an amine of the formulaNHR⁵ R⁶ where R⁵ and R⁶ are each hydrogen or lower alkyl, or byconversion of the tetrazole thiol where R² is hydroxy to thecorresponding acid chloride with subsequent reaction of the acidchloride with an amine (NHR⁵ R⁶). The tetrazole thiols of Formula IIIare also prepared by conversion of a suitably substituted hydroxytetrazole to the corresponding thiol by the method of Hoover and Day [J.Amer. Chem. Soc. 78:5832 (1956)].

The compounds of this invention are capable of forming salts with, forexample, the alkali metals such as sodium or potassium, the alkalineearth metals such as calcium or with the ammonium cation. When A is NH₂,the compounds can exist as the zwitterion or as an acid or base salt.These salts are prepared by standard methods using a wide variety ofnon-toxic pharmaceutically acceptable acids and bases known in the artand are also considered as objects of this invention.

It will be recognized that due to the asymmetric α-carbon atom in the7-acetamido group of Formula I when R³ is ##STR8## optical isomers willexist. Racemic or resolved products are obtained depending upon whethera racemic or resolved side chain acid is used as an acylating agent. Theresolved side chain acids are readily obtained from the racemiccompounds by resolution according to well known methods, includingfractional crystallization of a salt formed with an optically activeacid or base. All of the isomers, including separated isomers andmixtures thereof, are included within the scope of this invention.

The compounds of Formula I have antibacterial activity against bothGram-positive and Gram-negative organisms. Minimum inhibitoryconcentrations (MIC's) range from 0.2 to >200 μg./ml. in in vitrotesting. These results are shown in Table 1 below for representativecompounds of Formula I. In vivo mouse protection data are given in Table2. Compound names corresponding to numbers are given in the experimentalsection. For comparative purposes, data for cephamandole, which is7-mandelamido-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid, is also given in Tables 1 and 2.

                                      TABLE 1                                     __________________________________________________________________________    MIC (μg./ml.) in vitro                                                         S.         Strep.                   Sal-                                      aur-                                                                             S.     Fae-                      mo- Shi-                                                                             Entero.                                                                           Serra.                                                                            Entero.                                                                           Proteus                eus                                                                              aureus                                                                            S. calis                                                                             E. coli                                                                           E. coli                                                                           Kleb.                                                                              Kleb.                                                                              Pseu-                                                                             nella                                                                             gella                                                                            aerog.                                                                            marc.                                                                             cloacae                                                                           mor-               Com-                                                                              HH SK  villa-                                                                           HH  SK  HH  pneumo.                                                                            pneumo.                                                                            do. sp.                                                                           ATCC                                                                              HH ATCC                                                                              ATCC                                                                              HH  gani               pound                                                                             127                                                                              23390                                                                             luz                                                                              34358                                                                             12140                                                                             33779                                                                             SK 4200                                                                            SK 1200                                                                            HH 63                                                                             12176                                                                             117                                                                              13048                                                                             13880                                                                             31254                                                                             179                __________________________________________________________________________    I   6.3                                                                              3.1,                                                                              100                                                                              100 1.6 1.6,                                                                              1.6, 1.6, >200                                                                              1.6,                                                                              1.6,                                                                             6.3,                                                                              100,                                                                              0.8 3.1                       1.6            6.3 0.8  0.8      0.8 0.8                                                                              12.5                                                                              25                         II  12.5                                                                             12.5                                                                              200                                                                              100 3.1 12.5                                                                              1.6  6.3  >200                                                                              1.6 6.3                                                                              25  >200                                                                              1.6 >200               III 1.6                                                                              3.1 25 25  3.1 3.1 3.1  3.1  >200                                                                              0.8 3.1                                                                              3.1 25  3.1 3.1                IV  1.6                                                                              0.8 50 25  3.1 6.3 3.1  1.6  >200                                                                              3.1 0.8                                                                              6.3 100 1.6 3.1                V   1.6                                                                              3.1,                                                                              25 25  0.8 1.6 1.6  0.8, >200                                                                              0.8,                                                                              0.2,                                                                             1.6 12.5                                                                              0.8,                                                                              1.6,                      1.6                     0.4      0.4 0.4        0.4 3.1                VI  6.3                                                                              6.3 50 50  6.3 6.3 12.5 6.3  >200                                                                              1.6 3.1                                                                              6.3 100 3.1 50                 VII 3.1,                                                                             0.8,                                                                              6.3,                                                                             12.5,                                                                             0.4,                                                                              1.6,                                                                              0.8, 0.4, >200,                                                                             0.2,                                                                              0.2,                                                                             1.6,                                                                              6.3,                                                                              0.4,                                                                              1.6,                   1.6,                                                                             0.4,                                                                              6.3,                                                                             12.5,                                                                             0.4,                                                                              1.6,                                                                              0.4, 0.2, >200,                                                                             0.2,                                                                              0.2,                                                                             0.8,                                                                              6.3,                                                                              0.4,                                                                              0.8,                   0.8                                                                              0.4 1.6                                                                              25  0.8 3.1 0.8  0.8  >200                                                                              0.2 0.4                                                                              6.3 50  1.6 1.6                VIII                                                                              3.1,                                                                             0.2,                                                                              50,                                                                              50, 0.4,                                                                              3.1,                                                                              0.8, 0.4, >200,                                                                             0.8,                                                                              0.8,                                                                             3.1,                                                                              12.5,                                                                             0.8,                                                                              3.1,                   1.6                                                                              1.6,                                                                              3.1                                                                              100 1.6 1.6,                                                                              1.6, 1.6, >200                                                                              0.4 0.8                                                                              6.3,                                                                              50, 6.3,                                                                              6.3,                      6.3            6.3 1.6  0.8             100 100 3.1 1.6                IX  12.5                                                                             6.3 100                                                                              25  12.5                                                                              12.5                                                                              6.3  6.3  > 200                                                                             3.1 6.3                                                                              12.5                                                                              200 3.1 100                X   6.3                                                                              12.5                                                                              25 25  1.6 1.6 1.6  3.1  >200                                                                              0.8 1.6                                                                              3.1 25  1.6 25                 XI  3.1                                                                              3.1 50 50  0.4 3.1 0.8  0.8  >200                                                                              0.8 0.4                                                                              3.1 25  3.1 0.8                XII 3.1                                                                              0.4 12.5                                                                             12.5                                                                              1.6 1.6 1.6  0.8, >200                                                                              0.4,                                                                              0.4                                                                              3.1 12.5                                                                              0.8 1.6                                               1.6      0.8                                   XIII                                                                              3.1                                                                              0.8 12.5                                                                             25  1.6 3.1 0.8  1.6  >200                                                                              1.6 0.8                                                                              6.3 12.5                                                                              0.8 1.6                XIV 1.6                                                                              0.4 6.3                                                                              12.5                                                                              3.1 3.1 1.6  3.1  >200                                                                              3.1 0.8                                                                              6.3 25  1.6 1.6                XV  1.6                                                                              0.8 25 50  0.8 3.1 0.8  0.4  --  0.4 0.8                                                                              6.3 200 0.8 >200               XVI 0.4                                                                              0.8 6.3                                                                              12.5                                                                              0.4 1.6 0.8  0.4  >200                                                                              0.4 0.2                                                                              1.6 12.5                                                                              0.4 12.5               XVII                                                                              0.4                                                                              0.4 12.5                                                                             25  0.4 3.1 0.8  0.4  --  0.4 0.2                                                                              1.6 100 0.8 200                XVIII                                                                             3  3   100                                                                              100 0.8 6   0.4  0.4  --  0.8 3  13  >200                                                                              3   200                XIX 50 50  100                                                                              200 25  50  50   6    --  25  6  50  50  50  6                  XX  0.4                                                                              0.2 3.1                                                                              6.3 0.8 0.8 0.4  0.4  >200                                                                              0.2 0.2                                                                              3.1 12.5                                                                              0.8 12.5               cepha-                                                                            1.0                                                                              0.6 8  2.3 1.1 2.6 1.1  0.6  >200                                                                              0.6 0.2                                                                              2.9 13  0.7 1.3                man-                                                                          dole                                                                          __________________________________________________________________________

                  TABLE 2                                                         ______________________________________                                        ED.sub.50 in vivo (mg./kg.)                                                           E. coli 12140                                                                              Kleb. pneumo. 4200                                       Compound  s.c.     p.o.      s.c.    p.o.                                     ______________________________________                                        I         1.56     50        <0.78,0.14                                                                            25                                       II        0.86,1.56,                                                                             98,>200,  <0.78,0.26                                                                            13.2,15.5                                          5.5      200                                                        III       1.56     34        2.8     >50                                      IV        2.5      168       7.5     168                                      V         1.56     50        1.3     21.2                                     VI        <0.2,1.56                                                                              3,6.25    0.92    4                                        VII       0.86     25        0.78    17                                       VIII      0.86     50        0.46    25                                       IX        1.56     28.5      3.7     18                                       X         0.78     25        0.62    15.6,18.2                                XI        1.56     >50       0.37    25                                       XII       1.84     >50       2.9     --                                       XIII      1.56     --        0.54    --                                       XIV       1.1      --        1.1     --                                       XV        1.56     >50       1.12    46                                       XVI       0.78     25        0.54    --                                       XVII      0.28     6.25      0.78    15.5                                     XVIII     3.1      --        0.4     --                                       XIX       --       --        --      --                                       XX        0.70     21.5      --      --                                       cephamandole                                                                            <2,2     28        0.96    >50,50                                   ______________________________________                                    

In addition, the active compounds of this invention exhibit broadspectrum activity and show advantageously high blood serum levels andhalf-life values. It is also pointed out that the compounds of Formula Iwhere A is OH and X is phenyl are especially active against Serratia,Enterobacter and indole positive Proteus bacteria.

Pharmaceutical compositions having antibacterial activity which comprisea pharmaceutical carrier containing an active but non-toxic quantity ofa compound of Formula I as well as methods of combatting bacterialinfections by administering such a composition to an infected host in anontoxic amount sufficient to combat such infections are also objects ofthis invention. The administration may be orally or by parenteralinjection such as subcutaneously, intramuscularly or intravenously. Theinjection of suitably prepared sterile solutions or suspensionscontaining an effective, nontoxic amount of the new cephalosporincompound is the preferred route of administration.

The compounds of Formula I are formulated and administered in the samemanner as other cephalosporins. The dosage regimen comprisesadministration, preferably by injection, of an active but nontoxicquantity of a compound of Formula I selected from the dosage unit rangeof from 100 to 1000 mg. with the total daily dosage regimen being from400 mg. to 6 g. The precise dosages are dependent upon the age andweight of the subject and on the infection being treated and can bedetermined by those skilled in the art based on the data disclosedherein compared with that available to the art attained with knowncephalosporins.

The following examples illustrate the invention, but are not to beconstrued as limiting the scope thereof. Temperatures are in degreesCentigrade unless otherwise stated.

EXAMPLE 17-D-Mandelamido-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (I)

Glycine (60 g., 0.8 mol.) was added to a cooled (5°-10°) solution of89.6 g. (1.6 mol.) of potassium hydroxide in 200 ml. of water. Aftercomplete dissolution 60.8 g. (0.8 mol.) of carbon disulfide was addedand the reaction mixture was stirred at 25° for three hours. A solutionof 113.6 g. (0.8 mol.) of methyl iodide in 200 ml. of ethanol was addedwhile maintaining the temperature at 25°-30°. The reaction mixture wasstirred for two hours at 25°, then concentrated in vacuo and theremaining aqueous phase was basicified to pH 8.0 with aqueous sodiumcarbonate and extracted with ether. The aqueous phase was acidified topH 2.5 with dilute hydrochloric acid and extracted with ethyl acetate.The extract was concentrated in vacuo and the residue recrystallizedfrom toluene to give methyl carboxymethyldithiocarbamate.

A mixture of 16.5 g. (0.1 mol.) of methyl carboxymethyldithiocarbamateand 14.3 g. (0.22 mol.) of sodium azide in 150 ml. of water was heatedat 56° for 12 hours. The reaction mixture was extracted with ether, thenacidified to pH 1.5 with dilute hydrochloric acid and extracted withethyl acetate. The extract was dried (MgSO₄) and evaporated to drynessto give 1-carboxymethyltetrazole-5-thiol, m.p. 178°-179°.

1-Carboxymethyltetrazole-5-thiol was also prepared by refluxing amixture of 45.95 g. (0.316 mol.) of ethyl isothiocyanoacetate and 30.8g. (0.475 mol.) of sodium azide in 500 ml. of water for 2.75 hours.Ethyl acetate (400 ml.) was added to the cooled reaction mixture and itwas acidified to pH 1.9 with 3 N hydrochloric acid. The layers wereseparated, the aqueous phase was extracted three times with ethylacetate and the combined extracts were dried (MgSO₄) and evaporated todryness to give a residue which was chromatographed on silica gel with17:3:2 chloroform-isopropanol-formic acid to give the tetrazole thiol.

A mixture of 4.49 g. (10 mmol.) of 7-D-mandelamidocephalosporanic acidsodium salt, 2.40 g. (15 mmol.) of 1-carboxymethyltetrazole-5-thiol and2.52 g. (30 mmol.) of sodium bicarbonate in 40 ml. of water was heatedat 70° for four hours. The reaction mixture was cooled (ice bath) andacidified to pH 1.8 with 3 N hydrochloric acid. Ethyl acetate was added,the layers were separated and the organic phase was dried (MgSO₄). Etherwas added to the ethyl acetate solution, the solution was filtered,ether and petroleum ether were added to the filtrate and the resultingprecipitate was collected, decolorized in ethyl acetate solution andre-precipitated by addition of ether and petroleum ether to give thetitle compound.

The title compound was dissolved in methanol and the methanol solutionwas treated with 26 ml. of 0.196 N sodium methoxide in methanol. Themethanol was removed in vacuo and the residue was dissolved in a minimumamount of water to which isopropanol was added to give7-D-mandelamido-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid sodium salt.

C₁₉ H₁₇ N₆ O₇ S₂ Na.2H₂ O.C₃ H₈ O: Calculated: 42.30% C; 4.67% H; 13.45%N; Found: 42.67% C; 4.56% H; 13.39% N.

EXAMPLE 27-(D-α-Amino-4-hydroxyphenylacetamido)-3-(1-carboxymethyl-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (II)

A solution of 5.22 g. (10.0 mmol.) of7-(D-α-t-butoxycarbonylamino-4-hydroxyphenylacetamido)cephalosporanicacid and 2.40 g. (15.0 mmol.) of 1-carboxymethyltetrazole-5-thiol in 75ml. of pH 6.4 phosphate buffer solution was treated with sufficientsolid sodium bicarbonate to give a pH of 6.4. The mixture was heated at70° for 4.5 hours, cooled and chromatographed on silica gel with 15:5:2chloroform-isopropanol-formic acid as eluant to give7-(D-α-t-butoxycarbonylamino-4-hydroxyphenylacetamido)-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-(D-α-t-butoxycarbonylamino-4-hydroxyphenylacetamido)-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid was stirred at 25° with 25 ml. of trifluoroacetic acid and 25 ml.of 1,3-dimethoxybenzene for 1.25 hours. The mixture was evaporated todryness, ether was added to the residue and the precipitated salt wascollected and dissolved in 350 ml. of water containing a few drops oftrifluoroacetic acid. The aqueous solution was treated with excessAmberlite IR-45 ion-exchange resin to pH 2.7, then lyophilized to givethe title compound.

C₁₉ H₁₉ N₇ O₇ S₂.2H₂ O: Calculated: 41.70% C; 4.36% H; 17.22% N; Found:42.05% C; 4.08% H; 16.30% N.

EXAMPLE 37-D-Mandelamido-3-(1-N-methylcarbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (III)

A solution of 7.2 g. (44 mmol.) of 1,1-carbonyldiimidazole in 110 ml. ofdry tetrahydrofuran and 20 ml. of dimethylformamide was added to asolution of 7.0 g. (43.8 mmol.) of 1-carboxymethyltetrazole-5-thiol in110 ml. of dry tetrahydrofuran and 20 ml. of dimethylformamide.Tetrahydrofuran saturated with methyl amine was added and the reactionmixture was stirred at 25° for 12 hours. The mixture was concentrated,the residue was diluted with 200 ml. of water and the resulting solutionwas adjusted to pH 2-3 by addition of 3 N hydrochloric aid. The aqueoussolution was extracted with ethyl acetate and the extract was dried(MgSO₄) and evaporated to dryness. Ethyl acetate (15 ml.) and ether (10ml.) were added to the residue and it was cooled to inducecrystallization of 1-N-methylcarbamoylmethyltetrazole-5-thiol, m.p.137°-140°.

1-N-Methylcarbamoylmethyltetrazole-5-thiol (2.39 g., 10 mmol.) and 4.22g. (9.4 mmol.) of 7-D-mandelamidocephalosporanic acid sodium salt werereacted as described in the procedure of Example 1. After cooling, thereaction mixture was acidified to pH 4 with 3 N hydrochloric acid,refrigerated for 12 hours and extracted with ethyl acetate. The extractwas dried (Na₂ SO₄) and evaporated to dryness to give a residue whichwas dissolved in 5 ml. of absolute methanol and 15 ml. of ethyl acetate.To this solution was added 1 ml. of ethyl acetate containing 0.1 ml. ofcyclohexylamine and 100 ml. of ethyl acetate. The precipitate wascollected and dissolved in absolute methanol and the solution was addedto 300 ml. of ethyl acetate. The resulting precipitate was collected anddissolved in 70 ml. of water and 70 ml. of chloroform at pH 2. Thelayers were separated, the aqueous phase was extracted with ethylacetate and the combined extracts were dried (Na₂ SO₄) andrecrystallized from ethyl acetate-ether to give the title compound.

C₂₀ H₂₀ N₇ O₆ S₂.0.7C₄ H₈ O₂.0.5H₂ O: Calculated: 46.39% C; 4.71% H;16.61% N; Found: 46.78% C; 4.35% H; 16.36% N.

EXAMPLE 47-D-Mandelamido-3-(1-N,N-dimethylcarbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (IV)

To a solution of 5.42 g. (33.8 mmol.) of1-carboxymethyltetrazole-5-thiol in 75 ml. of dry tetrahydrofuran and 20ml. of dry dimethylformamide was slowly added a solution of 5.49 g.(33.9 mmol.) of 1,1-carbonyldiimidazole in 95 ml. of drydimethylformamide. The reaction mixture was stirred for 35 minutes, then250 ml. of tetrahydrofuran saturated with dimethylamine was added to thesuspension and it was stirred at 25° for 12 hours. The mixture wasconcentrated to about 200 ml. and tetrahydrofuran and ether were added.The precipitate was collected by filtration and dissolved in 170 ml. ofwater. The aqueous solution was acidified to pH 2.0 by addition of 6 Nsulfuric acid and extracted with ethyl acetate. The ethyl acetatesolution was evaporated to dryness and the residue was triturated withether containing 5% ethyl acetate to give1-N,N-dimethylcarbamoylmethyltetrazole-5-thiol, m.p. 190°-200° (dec.).

7-D-Mandelamidocephalosporanic acid sodium salt hydrate (3.59 g., 8mmol.) was added to a solution of 1.008 g. (12 mmol.) of sodiumbicarbonate and 2.244 g. (12 mmol.) of1-N,N-dimethylcarbamoylmethyltetrazol-5-thiol in 40 ml. of water and thereaction mixture was heated at 67° for four hours. After cooling, themixture was acidified to pH 5.0 with 3 N hydrochloric acid andrepeatedly extracted with ethyl acetate. The combined extracts weredried (MgSO₄) and evaporated to dryness to give the title compound.

The title compound was dissolved in methanol and the solution was addedto a mixture of 100 ml. of chloroform and 300 ml. of ether. Theresulting precipitate was collected and recrystallized from methanolcontaining cyclohexylamine to give the title compound as itscyclohexylamine salt.

C₂₁ H₂₃ N₇ O₆ S₂.1.5H₂ O.C₆ H₁₃ N: Calculated: 49.15% C; 5.95% H; 16.98%N; Found: 49.49% C; 5.38% H; 16.42% N.

EXAMPLE 57-D-Mandelamido-3-(1-carbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (V)

1-Carboxymethyltetrazole-5-thiol and 1,1-carbonyldiimidazole werereacted as described in Example 4. To the reaction mixture was addedtetrahydrofuran saturated with dry ammonia gas. The resulting suspensionwas stirred for 2.5 hours and the solid which formed was collected byfiltration, washed with tetrahydrofuran and dissolved in methanol.Amberlite IR-120 ion-exchange resin (50 g.) was added and the suspensionwas stirred for 15 minutes. The resin was then removed and washed withabsolute methanol. The methanol solution was concentrated to give aresidue which was dissolved in ether and triturated with acetone to give1-carbamoylmethyltetrazole-5-thiol, m.p. 200° (dec.).

1-Carbamoylmethyltetrazole-5-thiol (0.715 g., 4.5 mmol.) and 1.347 g.(3.0 mmol.) of 7-D-mandelamidocephalosporanic acid sodium salt werereacted as described in the procedure of Example 1. After cooling, thereaction mixture was acidified to pH 2.0 and extracted with ethylacetate until no product remained in the aqueous phase. The extractswere combined, dried (MgSO₄) and the solvent evaporated to give aresidue which was dissolved in 20 ml. of acetone and 10 ml. of benzene.Cyclohexylamine (1.2 mmol.) was added to the solution and theprecipitated salt was collected, washed with acetone-benzene (2:1) anddissolved in 40 ml. of water. The pH of the aqueous solution wasadjusted to 3 and the solution was extracted with ethyl acetate. Theextracts were dried (MgSO₄) and evaporated to dryness to give the titlecompound.

C₁₉ H₁₉ N₇ O₆ S₂.0.5H₂ O.1C₄ H₈ O₂ : Calculated: 45.84% C; 4.68% H;16.26% N; Found: 45.52% C; 4.34% H; 15.90% N.

EXAMPLE 67-(D-α-Amino-4-hydroxyphenylacetamido)-3-(1-carbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (VI)

A solution of 5.21 g. (0.01 mol.) of7-(D-α-t-butoxycarbonylamino-4-hydroxyphenylacetamido)cephalosporanicacid and 2.4 g. (0.015 mol.) of 1-carbamoylmethyltetrazole-5-thiol werereacted according to the procedure described in Example 2. Aftercooling, the reaction mixture was extracted with ethyl acetate. Freshethyl acetate was added to the aqueous phase and it was acidified withstirring to pH 2.8 with 6 N sulfuric acid. The layers were separated andthe aqueous phase was again extracted with ethyl acetate. The combinedextracts were washed with water, dried (MgSO₄) and the solventevaporated to give a solid which was recrystallized fromacetonechloroform and chromatographed on silica to give7-(D-α-t-butoxycarbonylamino-4-hydroxyphenylacetamido)-3-(1-carbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-(D-α-t-Butoxycarbonylamino-4-hydroxyphenylacetamido)-3-(1-carbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid was treated with trifluoroacetic acid as described in Example 2 togive the title compound.

C₁₉ H₂₀ N₈ O₆ S₂.3.5H₂ O: Calculated: 39.10% C; 4.66% H; 19.20% N;Found: 41.64% C; 4.39% H; 20.06% N.

EXAMPLE 77-D-Mandelamido-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid (VIII)

β-Alanine (17.8 g., 0.2 mol.) was added to a solution of 22.4 g. (0.4mol.) of potassium hydroxide in 500 ml. of water at 25°. Carbondisulfide (12.2 ml., 0.2 mol.) was added and the reaction mixture wasrefluxed for three hours. The mixture was cooled, 28.4 g. (0.2 mol.) ofmethyl iodide and 500 ml. of ethanol were added and the resultingmixture was stirred for 30 minutes. The precipitate was collected byfiltration, the filtrate was concentrated and the aqueous residue wascombined with the solid material and brought to pH 8.5-9 by addition of10% aqueous sodium hydroxide. The resulting suspension was extractedwith ethyl acetate. Ethyl acetate was added to the aqueous phase whichwas then acidified to pH 1.5 with 6 N hydrochloric acid. The layers wereseparated and the aqueous phase was extracted with ethyl acetate. Theethyl acetate extracts were combined, dried (MgSO₄) and evaporated todryness to give methyl 2-carboxyethyldithiocarbamate.

To a mixture of 25.37 g. (0.143 mol.) of methyl2-carboxyethyldithiocarbamate and 5.6 g. (0.143 mol.) of sodium hyroxidein 210 ml. of water was added 9.25 g. (0.143 mol.) of sodium azide. Thereaction mixture was refluxed for one hour then cooled, diluted with 100ml. of ether and acidified to pH 1.7. The layers were separated, theaqueous phase was extracted with ether and the combined extracts weredried (MgSO₄) and evaporated to dryness to give a residue which wasrecrystallized from acetone-chloroform to give1-(2-carboxyethyl)tetrazole-5-thiol, m.p. 158°-160°.

Substitution of an equivalent amount of1-(2-carboxyethyl)tetrazole-5-thiol in the procedure of Example 1 inplace of 1-carboxymethyltetrazole-5-thiol gave the title compound.

C₂₀ H₂₀ N₆ O₇ S₂.0.5H₂ O.0.1C₄ H₁₀ O: Calculated: 45.63% C; 4.13% H;15.65% N; Found: 45.82% C; 4.14% H; 14.22% N.

EXAMPLE 87-D-Mandelamido-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid (VII)

When an equivalent amount of 1-(2-carboxyethyl)tetrazole-5-thiol wassubstituted in the procedure of Example 5 for1-carboxymethyltetrazole-5-thiol, 1-(2-carbamoylethyl)tetrazole-5-thiolwas obtained, m.p. 181°-182° (dec.).

1-(2-Carbamoylethyl)tetrazole-5-thiol (1.73 g., 0.01 mol.) and 3.02 g.(6.7 mmol.) of 7-D-mandelamidocephalosporanic acid were reactedaccording to the procedure of Example 2 while maintaining the pH at 7.0by addition of sodium bicarbonate. The cooled solution was acidified topH 5.0 with 6 N sulfuric acid and then extracted with ethyl acetate. Theaqueous phase was acidified to pH 2.1 and extracted with ethyl acetate.The extract was washed with water, dried (MgSO₄) and evaporated todryness to give a residue which was chromatographed on silica to givethe title compound as a glassy solid which crystallized frommethanol-ether.

C₂₀ H₂₁ N₇ O₆ S₂.1.25H₂ O.0.2C₄ H₁₀ O: Calculated: 44.85% C; 4.61% H;17.60% N; Found: 45.30% C; 4.31% H; 16.98% N.

EXAMPLE 97-(D-α-Amino-4-hydroxyphenylacetamido)-3-(1-N,N-dimethylcarbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (IX)

A solution of 4.26 g. (8.18 mmol.) of7-(D-α-t-butoxycarbonylamino-4-hydroxyphenylacetamido)cephalosporanicacid and 2.3 g. (0.012 mol.) of1-N,N-dimethylcarbamoylmethyltetrazole-5-thiol were reacted according tothe procedure described in Example 2. After cooling, the reactionmixture was extracted with ethyl acetate. Fresh ethyl acetate was addedto the aqueous phase and it was acidified to pH 2.5 with 6 N sulfuricacid. The layers were separated and the aqueous phase was againextracted with ethyl acetate. The combined extracts were washed withwater, dried (Na₂ SO₄) and evaporated to dryness to give a residue whichwas chromatographed on silica to give7-(D-α-t-butoxycarbonylamino-4-hydroxyphenylacetamido)-3-(1-N,N-dimethylcarbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-(D-α-t-Butoxycarbonylamino-4-hydroxyphenylacetamido)-3-(1-N,N-dimethylcarbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid was treated with trifluoroacetic acid as described in Example 2 togive the title compound.

C₂₁ H₂₄ N₈ O₆ S₂.2.5H₂ O: Calculated: 42.86% C; 4.94% H; 18.85% N;Found: 43.05% C; 4.81% H; 17.65% N.

EXAMPLE 107-(D-α-Amino-4-hydroxyphenylacetamido)-3-[1-(2-carbamoylethyl)-tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid (X)

A mixture of 3.9 g. (0.022 mol.) of1-(2-carbamoylethyl)tetrazole-5-thiol and 7.82 g. (0.015 mol.) of7-(D-α-t-butoxycarbonylamino-4-hydroxyphenylacetamido)cephalosporanicacid was reacted by the procedure described in Example 2 with additionof sodium bicarbonate to give a pH of 6.8. After cooling, the reactionmixture was extracted with ethyl acetate. Fresh ethyl acetate was addedto the aqueous phase and it was acidified to pH 1.8. The layers wereseparated and the aqueous phase was again extracted with ethyl acetate.The extract was dried (MgSO₄) and evaporated to dryness to give aresidue which was chromatographed on silica gel with 7.5:2.4:1chloroform-ethanol-formic acid as eluant to give7-(D-α-t-butoxycarbonylamino-4-hydroxyphenylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

7-(D-α-t-Butoxycarbonylamino-4-hydroxyphenylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid was treated with trifluoroacetic acid as described in the procedureof Example 2 to give the title compound.

C₂₀ H₂₂ N₈ O₆ S₂.1.5H₂ O: Calculated: 42.77% C; 4.49% H; 19.95% N;Found: 42.70% C; 4.47% H; 19.06% N.

EXAMPLE 117-D-Mandelamido-3-[1-(3-carboxypropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid (XII)

When an equivalent amount of 4-aminobutyric acid was substituted in theprocedure of Example 7 for β-alanine, methyl3-carboxypropyldithiocarbamate was prepared.

Reaction of methyl 3-carboxypropyldithiocarbamate with sodium azide andsodium hydroxide as described in Example 7 gave1-(3-carboxypropyl)tetrazole-5-thiol, m.p. 99°-101°.

Substitution of an equivalent amount of1-(3-carboxypropyl)tetrazole-5-thiol in the procedure of Example 1 inplace of 1-carboxymethyltetrazole-5-thiol gave the title compound.

The title compound was converted to the corresponding sodium salt asdescribed in the procedure of Example 1.

C₂₁ H₂₁ N₆ O₇ S₂.Na.0.75H₂ O: Calculated: 44.25% C; 3.97% H; 14.74% N;Found: 43.99% C; 3.91% H; 14.71% N.

EXAMPLE 127-D-Mandelamido-3-[1-(3-carbamoylpropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid (XI)

When an equivalent amount of 1-(3-carboxypropyl)tetrazole-5-thiol wassubstituted in the procedure of Example 5 for1-carboxymethyltetrazole-5-thiol, 1-(3-carbamoylpropyl)tetrazole-5-thiolwas obtained, m.p. 133°-136°.

1-(3-Carbamoylpropyl)tetrazole-5-thiol (3.34 g., 18 mmol.) and 5.39 g.(12 mmol.) of 7-D-mandelamidocephalosporanic acid were reacted accordingto the procedure of Example 2 while maintaining the pH at 6.6 byaddition of sodium bicarbonate. The cooled reaction mixture wasacidified to pH 2.5 and extracted with ethyl acetate. The extract wasevaporated to dryness, the residue was precipitated from ethylacetate-ether and the precipitate was chromatographed on silica gel with8:2:1 chloroform-ethanol-formic acid to give the title compound.

C₂₁ H₂₃ N₇ O₆ S₂.2H₂ O: Calculated: 44.27% C; 4.77% H; 17.21% N; Found:44.46% C; 4.37% H; 16.87% N.

EXAMPLE 137-D-Mandelamido-3-[1-(5-carboxypentyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid (XIII)

Substitution of an equivalent amount of 6-aminocaproic acid in theprocedure of Example 7 for β-alanine gave methyl5-carboxypentyldithiocarbamate.

Reaction of methyl 5-carboxypentyldithiocarbamate with sodium azide andsodium hydroxide as described in Example 7 gave1-(5-carboxypentyl)tetrazole-5-thiol, m.p. 100°-100.5°.

Substitution of an equivalent amount of1-(5-carboxypentyl)-tetrazole-5-thiol in the procedure of Example 1 inplace of 1-carboxymethyltetrazole-5-thiol gave the title compound.

The title compound was converted to the corresponding sodium salt bytreatment with sodium methoxide as described in Example 1.

C₂₃ H₂₅ N₆ O₇ S₂.Na.0.75H₂ O: Calculated: 46.19% C; 4.47% H; 14.05% N;Found: 46.07% C; 4.48% H; 13.76% N.

EXAMPLE 147-D-Mandelamido-3-[1-(5-carbamoylpentyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid (XIV)

1-(5-Carboxypentyl)tetrazole-5-thiol (6.0 g., 28 mmol.) was slowlydissolved in 20 ml. of thionyl chloride and the mixture was stirred at25° for 1.5 hours. Evaporation of the reaction mixture to dryness gave aresidue which was dissolved in 30 ml. of tetrahydrofuran. Thetetrahydrofuran solution was added to a cold mixture of 60 ml. ofammonium hydroxide and 30 ml. of tetrahydrofuran and the resultingmixture was stirred at 25° for two days. The mixture was extracted withethyl acetate. The aqueous phase was acidified to pH 1 by addition of 6N hydrochloric acid to precipitate1-(5-carbamoylpentyl)tetrazole-5-thiol, m.p. 155°-157°.

1-(5-Carbamoylpentyl)tetrazole-5-thiol (1.6 g., 7.5 mmol.) and 1.79 g.(4 mmol.) of 7-D-mandelamidocephalosporanic acid were reacted accordingto the procedure of Example 2 while maintaining the pH at 6.8 byaddition of sodium bicarbonate. The cooled reaction mixture wasacidified to pH 2.5 and extracted with ethyl acetate. The extract wasevaporated to dryness and the residue was chromatographed on silica gelwith 7:3:1 chloroform-ethanol-formic acid as eluant to give the titlecompound.

The title compound was converted to the corresponding sodium salt asdescribed in Example 1 above.

C₂₃ H₂₆ N₇ O₆ S₂.Na.1.5H₂ O: Calculated: 45.24% C; 4.78% H; 16.01% N;Found: 45.31% C; 4.50% H; 15.62% N.

EXAMPLE 157-Trifluoromethylmercaptoacetamido-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid (XV)

To an aqueous solution of 2.12 g. (12.2 mmol.) of1-(2-carboxyethyl)tetrazole-5-thiol and 2.05 g. (24.4 mmol.) of sodiumbicarbonate in 35 ml. of water was added 3.66 g. (8.1 mmol.) of7-trifluoromethylmercaptoacetamidocephalosporanic acid. The reactionmixture was stirred at 70° for 4.5 hours, then cooled andchromatographed on XAD-2 resin with water and methanol as eluants. Themethanol solution was evaporated to dryness to give a residue which wasstirred with 30 ml. of water. Ethyl acetate was added and the resultingmixture was filtered and lyophilized to give7-trifluoromethylmercaptoacetamido-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid disodium salt.

C₁₅ H₁₃ F₃ N₆ O₆ S₃.2Na.1.5H₂ O: Calculated: 30.26% C; 2.75% H; 13.93%N; Found: 30.62% C; 2.73% H; 13.41% N.

7-Trifluoromethylmercaptoacetamido-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid disodium salt is dissolved in a minimum amount of water to whichchloroform is added. While stirring, 3 N hydrochloric acid is addeduntil the solution is acidified to pH 2.5. The layers are separated, theaqueous phase is extracted with chloroform and the combined extracts arewashed with water, dried (MgSO₄) and evaporated to dryness to give thetitle compound.

EXAMPLE 167-Trifluoromethylmercaptoacetamido-3-(1-carbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (XVII)

7-Trifluoromethylmercaptoacetamido-3-(1-carbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid sodium salt was prepared by reaction of 2.39 g. (15 mmol.) of1-carbamoylmethyltetrazole-5-thiol, 1.27 g. (15 mmol.) of sodiumbicarbonate and 4.52 g. (10 mmol.) of7-trifluoromethylmercaptoacetamidocephalosporanic acid by the proceduredescribed in Example 15.

C₁₄ H₁₃ F₃ N₇ O₅ S₃.Na.1.5H₂ O.0.1C₄ H₁₀ O: Calculated: 30.34% C; 3.00%H; 17.02% N; Found: 30.76% C; 2.45% H; 18.31% N.

The title compound is obtained from the salt as described in Example 15.

EXAMPLE 177-Amino-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

A solution of 1-(2-carbamoylethyl)tetrazole-5-thiol (10.4 g., 0.06 mol.)in 120 ml. of acetone was added to a warm (45°) solution of 10.9 g.(0.04 mol.) of 7-aminocephalosporanic acid in a mixture of 220 ml. ofwater, 50 ml. of acetone and 8.4 g. (0.01 mol.) of sodium bicarbonate.The temperature was raised to 65° and the pH maintained at 7.4-7.6 byaddition of aqueous sodium carbonate solution. After three hours, thereaction mixture was cooled to 10° and adjusted to pH 3.5 by addition ofdilute hydrochloric acid. The resulting solid was collected byfiltration, washed with water and acetone and suspended in 95 ml. of 1.5N hydrochloric acid. The acid suspension was stirred at 25° for fivehours, filtered and the pH of the filtrate was adjusted to 3.5 byaddition of solid sodium bicarbonate. The solid was collected byfiltration and washed with water and acetone to give the title compound.

EXAMPLE 187-Trifluoromethylmercaptoacetamido-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid (XVI)

A solution of 4.8 g. (0.03 mol.) of trifluoromethylmercaptoacetic acidand 3.45 g. (0.03 mol.) of N-hydroxysuccinimide in 50 ml. oftetrahydrofuran was stirred and cooled to 0° before 6.2 g. (0.031 mol.)of dicyclohexylcarbodiimide was added in one portion. The reactionmixture was stirred at 0° for one hour and then for 12 hours at 25°. Theprecipitate was collected by filtration and washed with tetrahydrofuran.The solid was dissolved in ether and the solution was decolorized withcharcoal and filtered. The filtrate was evaporated to give the activatedester of trifluoromethylmercaptoacetic acid.

A suspension of 3.85 g. (0.01 mol.) of7-amino-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid in 50 ml. of dry dimethylformamide was treated with 2 ml. oftriethylamine and the mixture was stirred for 15 minutes at 25°. Theactivated ester of trifluoromethylmercaptoacetic acid (2.57 g., 0.016mol.) was added to the mixture and it was stirred an additional hour.The reaction mixture was evaporated to dryness and water and ethylacetate were added to the residue. The layers were separated, ethylacetate was added to the aqueous phase and it was acidified to pH 2.5 byaddition of 6 N hydrochloric acid. The mixture was filtered, the layerswere separated and the aqueous phase was extracted with ethyl acetate.The extract was washed with water, dried (MgSO₄) and evaporated todryness to give the title compound.

The title compound was dissolved in 30 ml. of methanol and a 5% solutionof sodium methoxide in methanol was added until pH 6.9. Ether was addedand the resulting precipitate was collected and washed with ether togive7-trifluoromethylmercaptoacetamido-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid sodium salt.

C₁₅ H₁₅ F₃ N₇ O₅ S₃.Na: Calculated: 32.79% C; 2.75% H; 17.84% N; Found:32.52% C; 2.79% H; 17.54% N.

EXAMPLE 97-D-Mandelamido-3-[1-(10-carboxydecyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

A suspension of 56 g. (1.0 mol.) of potassium hydroxide and 100 g. (0.5mol.) of 11-aminoundecanoic acid in 170 ml. of water was stirred for 30minutes at 25° then 40 g. (0.52 mol.) of carbon disulfide and 80 ml. ofethanol were added and the reaction mixture was stirred at 25° for 12hours. The mixture was refluxed gently for two hours and cooled. Methyliodide (71 g., 0.3 mol.) and 130 ml. of ethanol were added to themixture and it was stirred at 25° for 12 hours. The mixture wasevaporated to remove the ethanol and the solid residue was collected byfiltration to give methyl 10-carboxydecyldithiocarbamate.

Methyl 10-carboxydecyldithiocarbamate (28 g., 0.096 mol.) was reactedwith 6.5 g. (0.1 mol.) of sodium azide according to the proceduredescribed in Example 7. Acidification upon work-up gave1-(10-carboxydecyl)tetrazole-5-thiol as a white precipitate, m.p.95°-98°.

1-(10-Carboxydecyl)tetrazole-5-thiol (4.29 g., 15 mmole.) was slowlyadded to a solution of 3.36 g. (40 mmol.) of sodium bicarbonate in 100ml. of water. Ethanol (30 ml.) was then added followed by 4.20 g. (10mmol.) of 7-D-mandelamidocephalosporanic acid and the mixture was heatedat 65° for 3.5 hours. Upon cooling, a precipitate formed which wascollected by filtration. The filtrate was extracted with ethyl acetate,the aqueous layer was acidified to pH 4 and extracted again with ethylacetate. The extract was dried (MgSO₄) and evaporated to dryness to givethe title compound.

The title compound was converted to the corresponding sodium salt asdescribed above.

C₂₈ H₃₅ N₆ O₇ S₂.Na: Calculated: 48.01% C; 5.71% H; 12.00% N; Found:48.42% C; 5.13% H; 11.35% N.

EXAMPLE 207-D-Mandelamido-3-[1-(10-carbamoyldecyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

1-(10-Carbamoyldecyl)tetrazole-5-thiol was prepared from1-(10-carboxydecyl)tetrazole-5-thiol by the procedure described inExample 5, m.p. 112°-114°.

Reaction of 1-(10-carbamoyldecyl)tetrazole-5-thiol and7-D-mandelamidocephalosporanic acid as described in Example 19 gave thetitle compound.

EXAMPLE 217-D-Mandelamido-3-[1-(2-carboxy-1-methylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

Substitution of an equivalent amount of 3-aminobutyric acid in theprocedure of Example 7 for β-alanine gave methyl(2-carboxy-1-methyl)ethyldithiocarbamate.

Treatment of methyl (2-carboxy-1-methyl)ethyldithiocarbamate with sodiumazide also as described in Example 7 gave1-(2-carboxy-1-methylethyl)tetrazole-5-thiol, m.p. 169°-172°.

7-D-Mandelamidocephalosporanic acid and1-(2-carboxy-1-methylethyl)tetrazole-5-thiol were reacted in thepresence of excess sodium bicarbonate as described in Example 2 to givethe title compound.

The title compound was converted to the corresponding sodium salt aspreviously described.

C₂₁ H₂₁ N₆ O₇ S₂.Na.2H₂ O: Calculated: 42.56% C; 4.22% H; 14.18% N;Found: 42.83% C; 3.88% H; 13.26% N.

EXAMPLE 22

When an equivalent amount of an amino acid listed below:

alanine

2-aminobutyric acid

2-aminovaleric acid

2-aminohexanoic acid

is used in the procedure of Example 7 in place of β-alanine and theresulting dithiocarbamates are treated with sodium azide as describedtherein, the following substituted tetrazole thiols are obtained:

1-(1-carboxyethyl)tetrazole-5-thiol

1-(1-carboxypropyl)tetrazole-5-thiol

1-(1-carboxybutyl)tetrazole-5-thiol

1-(1-carboxypentyl)tetrazole-5-thiol.

Reaction of a tetrazole thiol listed above with7-D-mandelamidocephalosporanic acid as described hereinabove gives thefollowing compounds of this invention:

7-D-mandelamido-3-[1-(1-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(1-carboxypropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(1-carboxybutyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(1-carboxypentyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

Likewise, reaction of a substituted tetrazole thiol listed above with7-aminocephalosporanic acid,7-(D-α-t-butoxycarbonyl-4-hydroxyphenylacetamido)cephalosporanic acid or7-trifluoromethylmercaptoacetamidocephalosporanic acid according to theprocedures described herein with subsequent removal of the protectivegroups as necessary, gives the corresponding7-amino-3-(carboxyalkyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacids,7-(D-α-amino-4-hydroxyphenylacetamido)-3-(carboxylalkyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacids and7-trifluoromethylmercaptoacetamido-3-(carboxyalkyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacids.

EXAMPLE 23

Reaction of an acid substituted tetrazole thiol listed in Examples 21 or22 with 1,1-carbonyldiimidazole and ammonia as described in theprocedure of Example 5 gives the following carbamoylalkyl substitutedtetrazole thiols:

1-(2-carbamoyl-1-methylethyl)tetrazole-5-thiol

1-(1-carbamoylethyl)tetrazole-5-thiol

1-(1-carbamoylpropyl)tetrazole-5-thiol

1-(1-carbamoylbutyl)tetrazole-5-thiol

1-(1-carbamoylpentyl)tetrazole-5-thiol.

Substitution of a tetrazole thiol listed above in the procedure ofExample 5 in place of 1-carbamoylmethyltetrazole-5-thiol gives thecephalosporin compounds of this invention listed below:

7-D-mandelamido-3-[1-(2-carbamoyl-1-methylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(1-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(1-carbamoylpropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(1-carbamoylbutyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(1-carbamoylpentyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

In a similar manner, 7-amino-, 7-(D-α-amino-4-hydroxyphenylacetamido)-and7-trifluoromethylmercaptoacetamido-3-(carbamoylalkyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacids are prepared from a substituted tetrazole thiol listed above and7-aminocephalosporanic acid,7-(D-α-t-butoxycarbonyl-4-hydroxyphenylacetamido)cephalosporanic acidand 7-trifluoromethylmercaptoacetamidocephalosporanic acid,respectively, with removal of the protective groups when necessary aspreviously described.

EXAMPLE 24

Reaction of the t-butoxycarbonyl derivative of the followingcephalosporanic acids:

7-(α-amino-4-formamidophenylacetamido)cephalosporanic acid

7-(α-amino-3-formamidophenylacetamido)cephalosporanic acid

7-(α-amino-4-ureidophenylacetamido)cephalosporanic acid

7-(α-amino-3-ureidophenylacetamido)cephalosporanic acid

7-(α-amino-4-hydroxymethylphenylacetamido)cephalosporanic acid

7-(α-amino-1,4-cyclohexadienylacetamido)cephalosporanic acid

7-(α-amino-3-fluoro-4-hydroxyphenylacetamido)cephalosporanic acid

with 1-(2-carbamoylethyl)tetrazole-5-thiol as described in the procedureof Example 2 followed by removal of the protective group as describedtherein gives the following compounds:

7-(α-amino-4-formamidophenylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-amino-3-formamidophenylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-amino-4-ureidophenylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-amino-3-ureidophenylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-amino-4-hydroxymethylphenylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-amino-1,4-cyclohexadienylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-amino-3-fluoro-4-hydroxyphenylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

By similar procedures described hereinabove, 7-(α-amino substitutedphenylacetamido)-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacids are prepared by reaction of the t-butoxycarbonyl derivative of thecephalosporanic acids listed above with1-(2-carboxyethyl)tetrazole-5-thiol followed by removal of theprotective group as previously described.

EXAMPLE 25

7-(4-Hydroxymandelamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid is prepared by reaction of 7-(4-hydroxymandelamido)cephalosporanicacid sodium salt and 1-(2-carbamoylethyl)tetrazole-5-thiol as describedin the procedure of Example 1.

Likewise, when 7-(4-hydroxymandelamido)cephalosporanic acid sodium saltand 1-(2-carboxyethyl)tetrazole-5-thiol are substituted in the procedureof Example 1 for 7-D-mandelamidocephalosporanic acid sodium salt and1-carboxymethyltetrazole-5-thiol,7-(4-hydroxymandelamido)-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid is obtained.

EXAMPLE 26

Reaction of acephalosporanic acid listed below:

7-(α-hydroxythienylacetamido)cephalosporanic acid

7-(α-carboxythienylacetamido)cephalosporanic acid

7-(α-sulphophenylacetamido)cephalosporanic acid with1-(2-carbamoylethyl)tetrazole-5-thiol or1-(2-carboxyethyl)tetrazole-5-thiol as described in the procedure ofExample 2 gives the following compounds of this invention:

7-(α-hydroxythienylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-hydroxythienylacetamido)-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-carboxythienylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-carboxythienylacetamido)-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-sulphophenylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-sulphophenylacetamido)-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 27

When the t-butoxycarbonyl derivative of7-(α-aminothienylacetamido)cephalosporanic acid is reacted with1-(2-carbamoylethyl)tetrazole-5-thiol or1-(2-carboxyethyl)tetrazole-5-thiol according to the procedure ofExample 2 followed by removal of the protective groups as describedabove,7-(α-aminothienylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid and7-(α-aminothienylacetamido)-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid are obtained, respectively.

EXAMPLE 28

A mixture of 2.5 g. (7.7 mmol). ofD-α-(N-t-butoxycarbonyl)-4-aminophenylglycine, 1.8 g. (9.2 mmol.) ofα-bromoacetic acid t-butyl ester and 2.5 g. (19.2 mmol.) of diisopropylethylamine in 15 ml. of ethanol was stirred at 25° for 48 hours. Thesolvent was removed in vacuo, the residue was diluted with ethyl acetateand sodium bicarbonate and the pH was adjusted to 2.5. The layers wereseparated and the aqueous phase was again extracted with ethyl acetate.The combined extracts were washed with saturated sodium chloridesolution, dried (MgSO₄) and evaporated to dryness to giveD-α-(N-t-butoxycarbonyl)-4-t-butoxycarbonylmethylaminophenylglycine.

A solution of 0.380 g. (1.0 mmol.) ofD-α-(N-t-butoxycarbonyl)-4-t-butoxycarbonylmethylaminophenylglycine,0.296 g. (1.0 mmol.) of 7-aminocephalosporanic acid t-butyl ester and0.210 g. (1.0 mmol.) of dicyclohexylcarbodiimide in 25 ml. of 9:1 ethylacetate-methylene chloride was stirred at 0° for one hour. The reactionmixture was filtered and the filtrate was washed with 2.5% sulfuricacid, 5% sodium bicarbonate and water, dried (MgSO₄) and evaporated todryness to give7-(D-α-t-butoxycarbonylamino-4-carboxymethylaminophenylacetamido)cephalosporanicacid t-butyl ester. Deblocking was accomplished by stirring a mixture ofthe cephalosporanic acid t-butyl ester and 2 ml. of benzenethiol in 10ml. of trifluoroacetic acid at 25° for one hour. Evaporation of thereaction mixture to dryness gave7-(D-α-amino-4-carboxymethylaminophenylacetamido)cephalosporanic acid.

Reaction of the t-butoxycarbonyl derivative of7-(D-α-amino-4-carboxymethylaminophenylacetamido)cephalosporanic acidwith 1-(2-carbamoylethyl)tetrazole-5-thiol or1-(2-carboxyethyl)tetrazole-5-thiol according to the procedure ofExample 2 with subsequent removal of the protective group as describedabove gives7-(D-α-amino-4-carboxymethylaminophenylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid and7-(D-α-amino-4-carboxymethylaminophenylacetamido)-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid, respectively.

EXAMPLE 29

When ethylamine, propylamine or butylamine is reacted with1-(2-carboxyethyl)tetrazole-5-thiol according to the procedure ofExample 3, the following tetrazole thiols are prepared:

1-(2-N-ethylcarbamoylethyl)tetrazole-5-thiol

1-(2-N-propylcarbamoylethyl)tetrazole-5-thiol

1-(2-N-butylcarbamoylethyl)tetrazole-5-thiol.

Reaction of a tetrazole thiol listed above with 7-aminocephalosporanicacid, 7-D-mandelamidocephalosporanic acid,7-(D-α-t-butoxycarbonylamino-4-hydroxyphenylacetamido)cephalosporanicacid and 7-trifluoromethylmercaptoacetamidocephalosporanic acid,respectively, with removal of the protective groups when necessary, asdescribed in Example 2, gives the following compounds of this invention:

7-amino-3-[1-(2-N-ethylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-amino-3-[1-(2-N-propylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-amino-3-[1-(2-N-butylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(2-N-ethylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(2-N-propylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(2-N-butylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(D-α-amino-4-hydroxyphenylacetamido)-3-[1-(2-N-ethylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(D-α-amino-4-hydroxyphenylacetamido)-3-[1-(2-N-propylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(D-α-amino-4-hydroxyphenylacetamido)-3-[1-(2-N-butylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-trifluoromethylmercaptoacetamido-3-[1-(2-N-ethylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-trifluoromethylmercaptoacetamido-3-[1-(2-N-propylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-trifluoromethylmercaptoacetamido-3-[1-(2-N-butylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 30

When diethylamine, dipropylamine or dibutylamine is substituted fordimethylamine in the procedure of Example 4, the following tetrazolethiols are prepared:

1-(2-N,N-diethylcarbamoylethyl)tetrazole-5-thiol

1-(2-N,N-dipropylcarbamoylethyl)tetrazole-5-thiol

1-(2-N,N-dibutylcarbamoylethyl)tetrazole-5-thiol.

Reaction of a tetrazole thiol listed above with 7-aminocephalosporanicacid, 7-D-mandelamidocephalosporanic acid,7-(D-α-t-butoxycarbonylamino-4-hydroxyphenylacetamido)cephalosporanicacid and 7-trifluoromethylmercaptoacetamidocephalosporanic acid,respectively, with removal of the protective groups when necessary, asdescribed in Example 2, gives the following compounds of this invention:

7-amino-3-[1-(2-N,N-diethylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-amino-3-[1-(2-N,N-dipropylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-amino-3-[1-(2-N,N-dibutylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(2-N,N-diethylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(2-N,N-dipropylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(2-N,N-dibutylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(D-α-amino-4-hydroxyphenylacetamido)-3-[1-(2-N,N-diethylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(D-α-amino-4-hydroxyphenylacetamido)-3-[1-(2-N,N-dipropylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(D-α-amino-4-hydroxyphenylacetamido)-3-[1-(2-N,N-dibutylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-trifluoromethylmercaptoacetamido-3-[1-(2-N,N-diethylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-trifluoromethylmercaptoacetamido-3-[1-(2-N,N-dipropylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-trifluoromethylmercaptoacetamido-3-[1-(2-N,N-dibutylcarbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 31

When a cephalosporanic acid listed below:

7-(3-sydnoneacetamido)cephalosporanic acid

7-cyanoacetamidocephalosporanic acid

7-(2-aminomethylphenylacetamido)cephalosporanic acid

is reacted with 1-(2-carbamoylethyl)tetrazole-5-thiol by the proceduredescribed in Example 2, the following compounds of this invention areobtained, respectively:

7-(3-sydnoneacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-cyanoacetamido-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(2-aminomethylphenylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

Similarly, reaction of a cephalosporanic acid listed above with1-(2-carboxyethyl)tetrazole-5-thiol as described in Example 2 gives thefollowing compounds of this invention:

7-(3-sydnoneacetamido)-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-cyanoacetamido-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(2-aminomethylphenylacetamido)-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 32

Reaction of a cephalosporanic acid listed below:

7-(2,2,2-trifluoroethylmercaptoacetamido)cephalosporanic acid

7-trifluoromethylsulfinylacetamidocephalosporanic acid

7-(4-pyridylthioacetamido)cephalosporanic acid

7-(3-pyridylthioacetamido)cephalosporanic acid

with 1-(2-carboxyethyl)tetrazole-5-thiol as described in the procedureof Example 15 gives the following compounds of this invention:

7-(2,2,2-trifluoroethylmercaptoacetamido)-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-trifluoromethylsulfinylacetamido-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(4-pyridylthioacetamido)-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(3-pyridylthioacetamido)-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 33

Reaction of a cephalosporanic acid listed in Example 32 with1-(2-carbamoylethyl)tetrazole-5-thiol by the procedure of Example 15gives the7-substituted-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacids listed below:

7-(2,2,2-trifluoroethylmercaptoacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-trifluoromethylsulfinylacetamido-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(4-pyridylthioacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(3-pyridylthioacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 347-(2,2,2-Trifluoroethylsulfinylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

The activated ester of 2,2,2-trifluoroethylsulfinylacetic acid isprepared as described in the procedure of Example 18. Reaction of theactivated ester with7-amino-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid as also described in Example 18 gives the title compound.

In like manner, the 7-(2,2,2-trifluoroethylsulfinylacetamido)derivatives of other 7-amino-3-substituted tetrazole cephalosporinsdescribed above may be prepared.

EXAMPLE 357-(2,2,2-Trifluoroethylsulfonylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

To a solution of 8.4 g. (0.019 mol.) of7-amino3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem4-carboxylicacid t-butyl ester and 3.9 g. (0.019 mol.) of2,2,2-trifluoroethylsulfonylacetic acid in tetrahydrofuran is addeddropwise a solution of 3.9 g. (0.019 mol.) of dicyclohexylcarbodiimidein 100 ml. of tetrahydrofuran. The reaction mixture is stirred at 25°for 12 hours, then filtered and concentrated to about 10 ml. The residueis filtered and evaporated to dryness to give7-(2,2,2-trifluoroethylsulfonylacetamido)-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid t-butyl ester.

The ester is dissolved in acetonitrile and trifluoroacetic acid isadded. The solution is stirred for three hours, then evaporated todryness to give the title compound.

Likewise, 7-(2,2,2-trifluoroethylsulfonylacetamido) derivatives of theother 7-amino-3-substituted tetrazole cephalosporins disclosed hereinare prepared.

EXAMPLE 367-Methylmercaptoacetamido-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

To a stirred, cooled (-20°) solution of 10.2 g. (0.026 mol.) of7-amino-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid in 220 ml. of 3% sodium bicarbonate and 220 ml. of acetone isdropwise added a solution of 3.66 g. (0.029 mol.) ofmethylmercaptoacetyl chloride in 52 ml. of acetone, during which timethe pH of the reaction mixture is maintained at 8.0 by addition of 10%sodium hydroxide. After addition the reaction mixture is stirred anadditional 20 minutes at -15°, then is warmed to 25° and extracted withether. The remaining aqueous phase is cooled, 250 ml. of ethyl acetateis added and the slurry is acidified with 3 N hydrochloric acid. Thelayers are separated and the aqueous phase is extracted twice more withethyl acetate. The combined extracts are dried (MgSO₄) and evaporated todryness to yield the title compound.

EXAMPLE 377-n-Propylmercaptoacetamido-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

The title compound is prepared by substitution of n-propylmercaptoacetylchloride in the procedure of Example 36 for methylmercaptoacetylchloride.

In a similar manner, other 7-alkylmercaptoacetamido-3-substitutedtetrazole cephalosporins of this invention are prepared from theappropriate 7-alkylmercaptoacetyl chloride and 7-amino-3-(substitutedtetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.

EXAMPLE 387-(α-Carboxythienylacetamido)-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (XIX)

7-Amino-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid was prepared as described in Example 17 from reaction of1-carboxymethyltetrazole-5-thiol and 7-aminocephalosporanic acid.

7-Amino-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (1.0 g., 2.68 mmol.) was suspended in 10 ml. of drydimethylformamide and 1.12 ml. of triethylamine was added followed by 85drops of formamide. A solution of 0.91 g. (2.68 mmol.) of the activatedester of α-t-butoxycarbonylthienylacetamide, prepared as describedabove, in 2 ml. of dimethylformamide was added and the reaction mixturewas stirred at 25° for three hours. An additional 0.45 g. of activatedester was then added and the mixture stirred another five hours. Ether(ca. 300 ml.) was added and the solution was decanted. The remainingoily material was washed with ether, dissolved in water and the aqueoussolution was extracted with ethyl acetate. The pH of the ethyl acetatesolution was adjusted to 1.5 by addition of dilute hydrochloric acid.Extraction with ethyl acetate and evaporation of the solvent gave7-(α-t-butoxycarbonylthienylacetamido)-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-(α-t-Butoxycarbonylthienylacetamido)-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (0.8 g.) was dissolved in a mixture of 10 ml. of trifluoroaceticacid and 10 ml. of m-dimethoxybenzene and stirred at 25° for one hour.Ether was added and the precipitate was collected and dissolved in 50ml. of ethyl acetate. Addition of sodium-2-ethylhexanoate gave7-(α-carboxythienylacetamido)-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid trisodium salt.

C₁₈ H₁₃ N₆ O₈ S₃.2.5Na.2H₂ O.0.15C₄ H₈ O₂ : Calculated: 34.62% C; 2.92%H; 13.02% N; Found: 35.34% C; 3.03% H; 12.26% N.

The salt was converted to the title compound as previously described.

EXAMPLE 39

When 7-aminoheptanoic acid or 9-aminononanoic acid is substituted in theprocedure of Example 19 in place of 11-aminoundecanoic acid and theresulting methyl carboxydithiocarbamate is treated with sodium azide asdescribed therein, 1-(6-carboxyhexyl)tetrazole-5-thiol and1-(8-carboxyoctyl)tetrazole-5-thiol are obtained, respectively.

Reaction of 1-(6-carboxyhexyl)tetrazole-5-thiol and1-(8-carboxyoctyl)tetrazole-5-thiol with 7-D-mandelamidocephalosporanicacid as described in Example 19 gives7-D-mandelamido-3-[1-(6-carboxyhexyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid and7-D-mandelamido-3-[1-(8-carboxyoctyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid, respectively.

EXAMPLE 40

1-(6-Carbamoylhexyl)tetrazole-5-thiol and1-(8-carbamoyloctyl)tetrazole-5-thiol are prepared from1-(6-carboxyhexyl)tetrazole-5-thiol and1-(8-carboxyoctyl)tetrazole-5-thiol according to the procedure describedin Example 5.

Reaction of 1-(6-carbamoylhexyl)tetrazole-5-thiol and1-(8-carbamoyloctyl)tetrazole-5-thiol with7-D-mandelamidocephalosporanic acid as described in Example 19 gives,respectively,7-D-mandelamido-3-[1-(6-carbamoylhexyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid and7-D-mandelamido-3-[1-(8-carbamoyloctyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 417-Trifluoromethylmercaptoacetamido-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (XVIII)

Reaction of 0.96 g. (6 mmol.) of 1-carboxymethyltetrazole-5-thiol and1.74 g. (4 mmol.) of 7-trifluoromethylmercaptoacetamidocephalosporanicacid as described in the procedure of Example 15 gave the title compoundas the corresponding disodium salt.

The disodium salt was converted to the title compound as previouslydescribed. The title compound was dissolved in ethyl acetate andcyclohexylamine and methanol were added to the solution. The mixture wasevaporated under reduced pressure to give the cyclohexylamine salt ofthe title compound.

C₁₃ H₁₁ F₃ N₆ S₃ O₆.C₆ H₁₃ N: Calculated: 39.15% C; 4.27% H; 15.98% N;Found: 39.36% C; 4.30% H; 15.75% N.

EXAMPLE 427-Trifluoromethylmercaptoacetamido-3-[1-(3-carbamoylpropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid (XX)

Reaction of 2.81 g. (15 mmol.) of1-(3-carbamoylpropyl)tetrazole-5-thiol, 4.52 g. (10 mmol.) of7-trifluoromethylmercaptoacetamidocephalosporanic acid and 1.27 g. (15mmol.) of sodium bicarbonate according to the procedure described inExample 15 gave the title compound as its sodium salt.

C₁₆ H₁₇ F₃ N₇ O₅ S₃.Na.0.75H₂ O: Calculated: 33.30% C; 3.23% H; 16.99%N; Found: 33.59%; C; 3.50% H; 16.44% N.

7-Trifluoromethylmercaptoacetamido-3-[1-(3-carbamoylpropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid sodium salt was converted to the title compound as described inExample 15.

EXAMPLE 43

An injectable pharmaceutical composition is formed by adding sterilewater or sterile saline solution (2 ml.) to 500 mg. of7-D-mandelamido-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid, sodium salt.

Pharmaceutical compositions of the other antibacterial compoundsdisclosed above may be formulated in a similar manner.

EXAMPLE 44

A tablet or capsule is formed from 500 mg. of7-trifluoromethylmercaptoacetamido-3-(1-carbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid, 250 mg. of lactose and 75 mg. of magnesium stearate.

Tablets or capsules of the other antibacterial compounds disclosed abovemay be formulated in a similar manner.

What is claimed is:
 1. A compound of the formula: ##STR9## in which:each individual R¹ is hydrogen or lower alkyl of from one to four carbonatoms;n is one to ten; R² is hydroxy, amino, lower alkylamino ordi(lower)alkylamino, each alkyl having from one to four carbon atoms;and R³ is an acyl group of the formula: ##STR10## where: Z is methyl,trifluoromethyl, trifluoroethyl or pyridyl; and m is zero to two,or anon-toxic pharmaceutically acceptable salt thereof.
 2. A compoundaccording to claim 1 in which R¹ is hydrogen and n is one to five.
 3. Acompound according to claim 2 in which Z is methyl, trifluoromethyl ortrifluoroethyl.
 4. A compound according to claim 3 in which R² ishydroxy or amino; Z is trifluoromethyl and m is zero.
 5. A compoundaccording to claim 4, said compound being7-trifluoromethylmercaptoacetamido-3-(1-carbamoylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.
 6. A compound according to claim 4, said compound being7-trifluoromethylmercaptoacetamido-3-[1-(2-carbamoylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.
 7. A compound according to claim 4, said compound being7-trifluoromethylmercaptoacetamido-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.
 8. A compound according to claim 4, said compound being7-trifluoromethylmercaptoacetamido-3-[1-(2-carboxyethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.
 9. A compound according to claim 4, said compound being7-trifluoromethylmercaptoacetamido-3-[1-(3-carbamoylpropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.
 10. A compound of the formula: ##STR11## wherein n is one to four.11. A compound of the formula: ##STR12## wherein n is one to four.